RESEARCH ARTICLE

Effect of combined microbes on plant toleranceto Zn–Pb contaminations

Anna Ogar1 & Łukasz Sobczyk2& Katarzyna Turnau1,3

Received: 15 May 2015 /Accepted: 16 July 2015 /Published online: 7 August 2015# The Author(s) 2015. This article is published with open access at Springerlink.com

Abstract The presence and composition of soil microbialcommunities has been shown to have a large impact onplant–plant interactions and consequently plant diversity andcomposition. The goal of the present study was to evaluateimpact of arbuscular mycorrhizal fungi (AMF) and nitrogen-fixing bacteria, which constitutes an essential link between thesoil and the plant’s roots. A greenhouse pot experiment wasconducted to evaluate the feasibility of using selected mi-crobes to improve Hieracium pilosella and Medicago sativagrowth on Zn–Pb-rich site. Results of studies revealed thatbiomass, the dry mass of shoots and roots, increased signifi-cantly when plants were inoculated with mycorrhizal fungiand nitrogen-fixing bacteria. The addition of Azospirillumsp. and Nostoc edaphicum without mycorrhiza suppressedplant growth. Single bacterial inoculation alone does not havea positive effect on M. sativa growth, while co-inoculationwith AMF improved plant growth. Plant vitality (expressed

by the performance index) was improved by the addition ofmicrobes. However, our results indicated that even dry heatsterilization of the substratum created imbalanced relation-ships between soil-plant and plants and associated microor-ganisms. The studies indicated that AMF and N2-fixers canimprove revegetation of heavy metal-rich industrial sites, ifthe selection of interacting symbionts is properly conducted.

Keywords Plant/microbial interactions . Arbuscularmycorrhizal fungi (AMF) . N2-fixing bacteria .

Cyanobacteria . Co-cropping . Plant vitality

Introduction

Environmental problems arising from tailings containingheavy metals are windblown dust dispersal, leaching of con-taminants into surface and groundwaters. Phytoremediationstrategies aims to decrease the environmental impact fromthe heavy metal laden waste by establishing vegetation cov-er over the degraded area. However, plant growth is ofteninhibited due to metal toxicity and a combination of factorsincluding low nutrient levels, acidity or alkalinity, poor wa-ter holding capacity, and poor physical structure. Unfavor-able air–water conditions lead to wind erosion in dry periodsas well as soil erosion during rainfall (Bradl 2004; Turnauet al. 2012). Plants and plant-associated microbes are in-volved in many biogeochemical processes operating in therhizosphere. Plants themselves alter soil chemistry throughchanges in pH and redox conditions (Alford et al. 2010).They also release various secondary metabolites includinginorganic and organic compounds that contribute to nutrientacquisition, accelerating metal mobility or immobilization(Bais et al. 2006; Toljander et al. 2007). Plants are naturallyassociated with microorganisms whose microbial

Responsible editor: Philippe Garrigues

Electronic supplementary material The online version of this article(doi:10.1007/s11356-015-5094-2) contains supplementary material,which is available to authorized users.

* Anna Ogaranna.ogar@uj.edu.pl

Łukasz Sobczyklukasz.sobczyk@uj.edu.pl

Katarzyna Turnaukatarzyna.turnau@uj.edu.pl

1 Plant-Microbial Interaction Research Group, Institute ofEnvironmental Sciences, Jagiellonian University, Krakow, Poland

2 Ecosystem Ecology Research Group, Institute of EnvironmentalSciences, Jagiellonian University, Krakow, Poland

3 The Malopolska Center of Biotechnology, Jagiellonian University,Krakow, Poland

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communities can directly or indirectly affect metal mobility,availability, and uptake of elements. Microbial consortia in-cluding mycorrhizal fungi and nitrogen-fixing bacteria couldfacilitate the survival of their host plants growing on metal-contaminated sites, by producing growth-stimulating sub-stances and/or by conferring increased tolerance to stress(Doornbos et al. 2012; Rajkumar et al. 2012). It has beenshown that co-inoculation with arbuscular mycorrhizal fungi(AMF) and nitrogen-fixing bacteria is a promising approachto favor the establishment and survival of legume plants inpoor soils (Tsimilli-Michael et al. 2000; Lin et al. 2007;Franzini et al. 2010). However, little is known about theeffects of mixed microbial consortia on the establishmentof legumes under heavy metal stress conditions. The aimof this study was to screen microbial interactions, whichwould take place in the rhizosphere of selected plant speciesgrown on Zn–Pb-rich substrate. Hieracium taxon was cho-sen as a model plant genus since it occurs in a large varietyof habitats and the genus have a worldwide distribution. Inopen environments, Hieracium pilosella shows vigorousclonal growth, resulting in the formation of dense mats.The leguminous species Medicago sativa has been sug-gested as a good candidate for remediation of metal-richtailings primarily due to its ability to fix atmospheric N2

and increase the pH of acid soils (Gardea-Torresdey et al.1998; Mar Vázquez et al. 2000; Turnau et al. 2012). Theremediation research has been carried out in Zn–PbTrzebionka tailing since 1990 (Turnau et al. 2012). Thestudy employed a full factorial experiment on H. pilosella,legume M. sativa, and microbial communities derived fromZn–Pb-rich tailing. Substrate sterilization procedures havebeen used to study the effect of introduced microbes onplant growth and to eliminate the influence of other soilmicrobial communities including soil-borne plant pathogens.Plants were inoculated with combinations of selected mi-crobes such as Rhizophagus irregularis (syn. Glomusintraradices) and indigenous strains of Azospirillum sp.and Nostoc edaphicum. The concept of single, dual, and/ormultilevel co-inoculations was studied in relation to plantperformance. The study was performed to (1) assess theeffectiveness of microbial inoculation and co-cropping inthe formation of a more stable vegetation cover on Zn–Pbtailings, (2) determine interaction between plants and micro-bial consortia, (3) determine possible interactions of co-cropped plants: alfalfa (M. sativa) and hawkweed(H. pilosella) growing together on contaminated site, (4)check whether microbial inoculation could be useful in es-tablishment of plants on the Zn–Pb-rich substratum, (5) se-lect the most efficient combination of microbial inoculationto improve plant growth on tailing, and (6) compare theeffect of microbial inoculation on growth and photosyntheticparameters of plants grown on non-sterilized and dry heatsterilized tailing.

Materials and methods

Site characterization

The ZG Trzebionka Mine Company is located near Chrzanów(Southern Poland, 30 km west of Kraków, N 50° 09′, E 19°25′). The Chrzanów district has a long history of metal oremining. During the twentieth century, extraction of Zn–Pbores was carried out by the ZG Trzebionka Mine Company.The Trzebionka ore field is located in the SE part of the Sile-sian–Cracow ore district. Zinc and lead ore were extractedfrom Mississippi Valley-type mines where deposits were lo-cated in Triassic dolomites. These are strata-bound tabular orebodies with very complex internal structures. The primary oreminerals are sphalerite (ZnS) and galena (PbS) accompaniedby iron sulfides, cerussite, smithsonite, and hemimorphite.The wall rock is crystalline dolostone, called Bore-bearingdolomite.^ The ore grade is low: 4.2 % Zn and 1.7 % Pb (onaverage) (Mucha and Szuwarzyński 2004). Since 1970, exca-vated ores have been subjected to flotation processes. Thewaste material produced as a by-product of flotation was de-posited 1 km from the ore extraction site over ca. 40 years andresulted in the formation of a 60 m high and 64 ha area oftailing. Each year, 2.2 million tons of ores were extracted(Szuwarzyński 1993). Extraction activity was terminated in2009 when resources were exhausted. The slopes were con-structed from the coarser grained fractions of wastes, separat-ed in hydrocyclones (Turnau et al. 2012). The ponds are now adanger to the environment because of the eolian erosion takingplace on the surrounding slopes and on the dried out portion ofthe plateaus above the slopes. Low porosity results in unfa-vorable air–water conditions, restricted water infiltration dur-ing rainfall, and restricted water recharge by capillary risefrom deeper layers during dry periods. These conditions favorwind erosion in dry periods and water erosion during rainfall(Trafas 1996). The list of vascular plant species recorded onZG Trzebionka zinc wastes were presented at Turnau et al.2012.

Waste material characterization

The chemical composition of the tailing material is unfavor-able for plant growth because the carbonate content exceeds75 %. High concentrations of Ca2+ and SO4

2− ions togetherwith low concentrations of Na+, K+, Mg2+, Cl−, and HCO3

−

ions are also typical for this site. Original tailing materialcontains no organic matter and is P- and N-deficient. ThepH of the waste ranges from 7 to 8. Growth substratum fromthe Zn–Pb tailing contained high concentrations of heavymetals: 468 μg g−1 cadmium, 7068 μg g−1 lead, and 53,303 μg g−1 zinc. The analysis of metal content in substratumwas done using total reflection X-ray fluorescence (TXRF)

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(Turnau et al. 2008). The soil moisture ranged from 0.06 to0.15 m3 m−3 (Ryszka and Turnau 2007).

AMF inoculation

The AMF inocula used was R. irregularis UNIJAG PL. 30/BR 1 obtained from the AMF collection (Jagiellonian Univer-sity, Kraków). R. irregulariswas propagated on stock cultureswith Zea mays for 6 months. Colonized root fragments, my-celium, and a sand–soil mixture containing propagules (ca.100 propagules g−1 of substratum) were used as inoculum.

Isolations of plant growth-promoting rhizobacteria

Both associated bacteria (Azospirillum sp. and N. edaphicum)were isolated from native ecotype ofH. pilosella andM. sativaroots/rhizosphere that were collected at the Zn–Pb-rich tailing.The bacterial strains were isolated following protocols forPGPR isolation (Bashan et al. 1993). Fifteen strains ofrhizobacteria and five strains of diazotrophic bacteria wereobtained after isolation. In in vitro cultures, plants weresubcultured with bacterial strains on the modified Strullu–Romand medium (MSR) (Promega Benelux, Leiden,The Netherlands). Cultures were cultivated for 2 months in agrowth chamber at 24 °C, under a 12/12 h light regime. Twostrains of N2-fixers: Azospirillum sp. and N. edaphicum wereselected for further examination. Taxonomic identificationwas based on cell/colony morphologies using the followingreferences: (Starmach 1966; Ettl and Gärtner 1995; Hoek1995; Hindák 1996; Jeffery et al. 2010).

Azospirillum sp.

The bacterial inoculum of Azospirillum sp. was grown onnutrient broth (Difco Bacto, USA). Flasks were incubated at26 °C, for 36 h on a rotary shaker (170 rpm). Actively growingcells were then washed three times with sterile phosphate-buffered saline (100 mM phosphate buffer; 0.85 % NaCl;pH 7.0) by centrifugation (10 min, 15,000×g) (Murty andLadha 1988). The washed cells were resuspended in the samebuffered saline as described earlier to a final concentration ofabout 108 colony-forming units (cfu)/ml. Bacterial suspensionwith an optical density at 600 nm (OD600) corresponded to afinal concentration of ∼108 bacteria/ml. Twenty milliliters ofthe bacterial inoculation suspensions was poured onto thesubstrate surface of each pot to initiate infection and subse-quent nodulation. Control treatments were inoculated withsterilized (121 °C, 20 min, 1 bar) inoculation suspension.

Nostoc edaphicum Kondrateva

Cyanobacteria, N. edaphium, were grown in 250-ml Erlen-meyer flasks containing 100 ml of Jaworski medium at pH

7.0. The flasks were incubated at 26 °C, for 3 weeks on arotary shaker (120 rpm). Actively growing cells were harvest-ed by centrifugation (10 min, 15,000×g) then washed threetimes with sterile physiological saline (9 g l−1 NaCl) and re-suspended in sterile. Treatments without cyanobacteria weretreated in the same way with sterilized (121 °C, 20 min, 1 bar)inoculation suspension. Twenty milliliters of the bacterial in-oculation suspensions was poured onto the substrate surfaceof each pot.

Experimental design

A greenhouse experiment utilizing a full factorial randomizedblock design was implemented. The experiment was conduct-ed with the use of two plant species (H. pilosella L. andM. sativa L.). H. pilosella was chosen as a model plant basedon observations made on ZG Trzebionka site where this plantgrows vigorously. Its main way of spreading is clonal growth.It usually formed new seedlings on the top of the partly driedtufts, and the new ramets were formed outside. Such rametssometimes disappeared while it was hot and dry, but after therain, they were usually rebuilt from the remaining parts. In theplaces where H. pilosella appeared in the following seasons,some other accompanying plants established, such asM. sativa. The growth of M. sativa was nearby H. pilosellapatches, very rarely growing alone on this particular tailing.Due to N2-fixing abilities,Medicago species were proposed asgood candidates for remediation strategies to enrich poor innutrient substrata. There were several attempts to introduceM. sativa, but this plant was not able to survive there. There-fore, an experiment under laboratory conditions on zinc–leadwaste was carried out to discover if the growth of alfalfa canbe improved by the co-cropping and introduction of mycor-rhizal fungi and N2-fixing bacteria.

Control plants H. pilosella (Hp) and M. sativa (Ms) weregrown separately, and they were also co-cropped (Hp + Ms).No inoculation was provided for control plants. Eight differentinocula variant were applied: (1) AMF; (2) Azospiriullum sp.;(3) Azospiriullum sp. + AMF; (4) Nostoc edaphicum; (6)N. edaphicum + AMF; (7) Azospirillum sp. + N. edaphicum;(8) Azospiriullum sp. + N. edaphicum + AMF. Plants weregrown on the Zn–Pb-rich tailing. Experiment was conductedon the non-sterile (NS), where no treatments were provided,and on the sterile substratum (S), where dry heat sterilizationwas applied to sterilized substratum. Waste material was ster-ilized at 100 °C for 2 h, over 3 days in a row and then allowedto cool for 72 h to eliminate biotic communities, but still retainabiotic tailing traits. Next, the plants were inoculated witharbuscular mycorrhizal fungi R. irregularis (M), Azospirillumsp. (A), andN. edaphicum (N) in different combinations or leftnon-inoculated (controls). Each combination was replicatedfive times for a total of 100 pots. Two compartmented culti-vation systems with 37-μm polyester mesh (Sefar LFM,

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Switzerland) were provided to separate H. pilosella andM. sativa roots. Seeds of H. pilosella ecotype were obtainedfrom populations grown at an industrial waste disposal areaZG Trzebionka. Seeds of M. sativa (V29814/04/001) wereobtained from Malopolska Hodowla Roslin (HBP, Poland,Krakow). Both types of seeds were surface sterilized with(1 % chloramine T (3 min), 6 % sodium hypochlorite(2 min), and 90 % ethanol (2 min)) and washed three timeswith distilled sterile water between each step. Seeds ofH. pilosella were pregerminated on 3 % water agar undergreenhouse conditions. Fourteen-day-old seedlings weretransferred into plastic pots (14 cm tall by 11 cm wide) andfilled with 500 g of tailing material. Each pot was sown with100 seeds of Alfalfa. Plants were watered three times a weekwith deionized water. Pots were regularly weighed tomaintainmoisture content at 80% ofwater-holding capacity. The plantswere not supplied with any nutrient solution. The experimentwas conducted in a controlled environment in growth chamber(PaNELTECH, Poland with an in-built TAC Xenta/Vista sys-tem) maintained at 24 °C, under a 12/12 h light regime. Max-imum photosynthetic photon flux (PPF) was 110±10 μmol(s m2)−1, and supplementary light was not needed.Relative humidity ranged from 20 to 25 %. Plants were har-vested after 17 weeks of growth.

Plant vitality

Plant vitality was evaluated before harvesting using a PlantEfficiency Analyzer (Hansatech Instruments, UK) estimatingchlorophyll a fluorescence transient of intact leaves. Measure-ments were taken on the upper surface of fully expandedleaves. For each soil/microbe combination, three leaves ofeach H. pilosella plant and 15 M. sativa plants per pot weremeasured. The collected data set was used for the JIP testanalysis (analysis of O-J-I-P fluorescence transient) (Strasserand Srivastava 1995; Tsimilli-Michael et al. 2000; Strasseret al. 2004). Although indirect, the JIP test allows informationabout the structure and function of the photosynthetic appara-tus (mostly related to PSII) to be obtained. The photosyntheticefficiencies, i.e., the maximum quantum yield of PSIIat t=0,φPo=TR0/ABS=1− (Fo/Fm)=Fv/Fm was measured. Thespecific flux parameters chosen to be calculated in the presentstudy, all referring to the condition of the sample at time zero,expressed per reaction center (RC) were analyzed: (1) ABS/RC—the average absorption per RC; (2) TR0/RC—the spe-cific trapping flux per RC; (3) DI0/RC—the dissipated energyflux per RC; (4) ET0/RC—the maximal specific flux for elec-tron transport per RC. Parameters were derived from the the-ory of energy flux from biomembranes (Strasser et al. 2000,2004). Also, the performance indexes which are products ofterms expressing Bpotentials^ for photosynthetic performancewhere PIABS: performance index (PI) on absorption basisPIABS=[RC/ABS] [TR0/(ABS−TR0] [ET0/(TR0−ET0)] and

PITOTAL: total PI, measuring the performance up to the PSIend electron acceptors, PITOTAL=PIABS [RE0/ET0−RE0)]were analyzed. The logarithms of performance indexes at t=0 log (PITOTAL, PIABS) were defined as the total driving force(DFTOTAL, DFABS) for photosynthesis of the observed system,created by summing the partial driving forces for each ofseveral energy bifurcations (Strasser et al. 2004).

Harvest and sample preparation

Each plant was carefully removed from the pot. Loosely ad-hering waste material was removed from the roots, and largerroot pieces remaining in the substrate were manually pickedout. Root and aerial part biomass of harvested plant wasweighted separately. Total fresh biomass of roots was weight-ed; after it, one fourth of roots was taken for staining to esti-mate mycorrhizal colonization. Collected biomass was frozenat −20 °C and then freeze-dried for 72 h, at −55 °C (ChristBeta 1–8 LD plus, SciQuip Ltd., UK). Dry weights of shootsand roots were determined after the freeze-dried procedure.

Arbuscular mycorrhizal colonization

At harvest, roots were removed from substrate and then gentlyrinsed with running tap water and then with deionized water(DI). Mycorrhizal root parts were randomly collected andcleared in 10 % KOH for 24 h at room temperature. Subse-quently, after careful washing in tap water, the roots wereacidified for 1 h in 5 % lactic acid and stained for 24 h at roomtemperature in 0.05 % aniline blue in lactic acid, in order tovisualize the fungal structures inside the roots. Mycorrhizalcolonization was estimated in the roots of H. pilosella andM. sativa. Root colonization by R. irregulariswas determinedaccording to protocol provided in Mycorrhiza Manual(www2.dijon.inra.fr/mychintec/Protocole/Workshop_Procedures.html). Since experiment was carried out in plantgrowth chamber, the possibilities of contamination byairborne spores from other AMF was limited. The followingparameters were assessed: intensity of the mycorrhizalcolonization in the root system (M%), intensity, andarbuscule abundance in the root system (A%). From eachpot, approximately 80 cm of fine root fragments wererandomly taken for mycorrhizal estimation. Mycorrhizalcolonization and arbuscular richness were estimatedmicroscopically in about 60 1-cm-long root fragments per pot.

Medicago sativa nodules

Roots were shaken gently to remove substrate, then washedgently and dried with moist tissue. Nodules were removed andfixed in formaldehyde–acetic acid–ethanol (FAA). The num-ber of nodules was counted per pot. Different nodules: white,pink, brown, hard, and soft were collected form roots.

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Statistical analysis

For biomass, dry mass, and mycorrhizal parameters, meanswere compared using ANOVAThe Tukey’s test was used asa post hoc test when ANOVA showed significance. If assump-tions were not met, we used non-parametric tests (Kruskal–Wallis test).

For photosynthetic parameters, first was performed theprincipal component analysis (PCA) to select variables withthe highest loadings for first and second axis. Then, for select-ed variables, ANOVA analysis was performed. If assumptionswere not met, we used non-parametric tests (Kruskal–Wallistest). For PCA, we used CANOCO 5 software (Ter Braak andŠmilauer 2012). For ANOVA and other tests, we usedSTATISTICA (version 10 StatSoft, 2010) software.

Results

Biomass production

Plants grown (M. sativa and H. pilosella) in NS Zn–Pb-richtailing demonstrated stunted growth consistent with S substra-tum in comparison with plants grown onNS substrate (Fig. 1).Significant differences between microbial treatments regard-ing biomass were observed. Growth of H. pilosella was sig-nificantly lower in the dry heat sterilized tailing material thanon the non-sterile substratum. Significantly higher (p<0.05)shoots biomass/dry mass was observed whenH. pilosellawasinoculated with microbes. Especially when plants were grownon sterile substrate, AMF inoculation together withdiazotrophs: Azospirillum sp. and N. edaphicum exerted apositive effect on Hieracium growth (Fig. 1a). No significantchanges of H. pilosella biomass were observed in the non-sterile substrate compared to the control plants (Fig. 1a). Al-though, stimulation ofH. pilosella growth was observed whenplants were co-inoculated with AMF and N2-fixers whilecompared to single inoculation with N2-fixers (Nostoc orAzospirillum) (Fig. 1a). Similar trends were also obtained forroot biomass and dry weights (data not shown).

Mycorrhizal inoculation together with N2-fixers alwaysstimulated alfalfa growth, while single and double inoculationwith Azospirillum sp. or N. edaphicum did not have a positiveeffect on M. sativa shoot growth (Fig. 1b). In S substrate,single inoculation with Azosporillum sp. was as high as inthe case of control plants (Fig. 1b). Biomass of M. sativa

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�Fig. 1 Biomass in (g) of a Hieracium pilosella shoots (Hp), bMedicagosativa shoots (Ms), c Medicago sativa roots; plants grown on NS non-sterilized substrate, S dry heat sterilized substrate, and inoculated withdifferent microorganisms: M Rhizophagus irregularis, A Azospirillumsp., N Nostoc edaphicum; different letters above bars indicatestatistically significant differences (P<0.05)

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grown on both (S) and (NS) substrates increased, when plantswere inoculated with R. irregularis. On sterile substrate, pos-itive effect of mycorrhizal inoculation was even more pro-nounced (Fig. 1b). Root growth of H. pilosella was greatlyreduced when plants were supplied with a single inoculationwith a N. edaphicum in NS treatment and double inoculatedwith both N2-fixers in S substrate (Fig. 1c).

To conclude, growth of non-inoculated plants in bothsubstrates (S and NS variant) was reduced. Dry heat ster-ilization procedure had the greatest effect, reducing shootsand root growth of both plant species. In general, shootbiomasses as well as dry weights (not shown) ofH. pilosella and M. sativa were higher in non-sterile sub-strate than in sterile one (Fig. 1a, b). Always, presence ofAMF positively affected fresh biomass of both testedplant species (p<0.05). There was significant interactionbetween sterilization procedure and various microbialtreatments for H. pilosella. Co-inoculation (AMF + N2-fixers) was effective and increased productivity ofH. pilosella grown on the Trzebionka tailing. The greatestM. sativa shoot development was reached in plants duallyinoculated with R. irregularis + N. edaphicum on bothsubstratum (S and NS). Non-inoculated control plantsand inoculated plants did not show any differences in rootgrowth except inoculation with N. edaphicum (on non-sterile substrate) and double N2-fixer inoculation (on ster-ile substrate), which negatively affects alfalfa root growth(Fig. 1c).

Root AMF colonization

Intensity of the mycorrhizal colonization in the root system(M%)

Roots of inoculated plants were extensively colonized byR. irregularis, and percentage root colonization ofM. sativa was comparable with that of H. pilosella. Dryheat sterilization procedure caused decreases of mycorrhi-zal colonization of both plants (Fig. 2a, b). In the case ofM. sativa, no statistically important interactions werefound between substratum treatment and inoculation op-tion, while significant interactions were observed forH. pilosella. The most efficient treatments were thosewhen H. pilosella was co-inoculated with Azospirillumsp. as well as when double inoculation with both N2-fixerswas provided (Fig. 2a) . Alfalfa inoculated withN. edaphicum (N) and R. irregularis (M) grown on sterileas well as on non-sterile substratum were significantly dif-ferent from all other treatments (Fig. 2b). Addition ofN. edaphicum significantly decreased intensity of the my-corrhizal colonization in the root system of both plant spe-cies whether plants were grown on sterilized substrate ornot (Fig. 2a, b).

Arbuscular richness: arbuscule abundance in the root system(A%)

On sterilized substrate, mycorrhizal colonization was signifi-cantly lower. Significant decreases in A% values were ob-served for both plant species. Arbuscule richness in root frag-ments where the arbuscules were abundant (A%) showed im-portant differences between substrate sterilization treatmentand microbial inoculation variant for H. pilosella species.When H. pilosella was grown on non-sterile substrate, thehighest rate of arbuscular richness was observed for plantsinoculated with both N2-fixers (Fig. 3a). While, single inocu-lation of H. pilosella with cyanobacteria, as well as double

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Fig. 2 Relative mycorrhizal root length (M%) evaluated for aHieraciumpilosella (Hp), b Medicago sativa (Ms), when plants were grown on NSnon-sterilized substrate, S dry heat sterilized substrate; plants wereinoculated with M Rhizophagus irregularis, A Azospirillum sp., NNostoc edaphicum; different letters beside the values indicatestatistically significant difference (P<0.05)

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with N. edaphicum and Azospiruillum sp. negatively affectedarbuscule abundance (A%) in the root system on sterile sub-strate (Fig. 3a). The arbuscule abundance in the alfalfa rootsystem (A%) was significantly higher when plants weregrown on non-sterile substrate when compared to sterile sys-tem (Fig. 3b). Inoculation with N2-fixers caused decrease inthe arbuscule richness in root fragments where the arbusculeswere abundant (A%) in the M. sativa root system (Fig. 3b).However, in non-sterile substrate, other microbes present inthe substrate probably attenuated negative effect of N2-fixerson arbuscular richness. Sterilization procedure of the substra-tum prior to decreased mycorrhizal colonization. Inoculationwith N2-fixers affected the percentage of AMF colonization.

The response of H. pilosella was different depending on ster-ilization procedure of the substrate. When plants were grownon non-sterile substrate, nitrogen-fixing microbial inoculantspositively influenced H. pilosella root colonization, while onsterile substrate, N2-fixers negatively affected arbuscule abun-dance (Fig. 3a). The lowest survival rate and lowest arbusculeabundance that were found for both plants were single inocu-lation with N. edaphicum was applied (Fig. 3a, b).

Nodule number

The number of nodules in alfalfa inoculatedwithR. irregularisgrown on non-sterile were significantly higher, whilst a veryfew nodules were observed in sterile treatments (Fig. 4). Abeneficial effect of applied microbial inoculations was foundfor both S and NS substrates (Fig. 4). The +AMF treatmentssignificantly enhanced the development of root nodules. Dualinoculation with the R. irregularis and the associateddiazotrophs (Azospirillum sp. andN. edaphicum) also resultedin a synergistically increased nodule number. The highest nod-ule number on non-sterile substrate was found when both N2-fixers and AMF was applied (Fig. 4). When M. sativa wasgrown on dry heat sterilized substrate, nodules were formedjust when plants were co-inoculated with mycorrhiza and N2-fixing bacteria. Otherwise, nodules where not present (Fig. 4).

Plant vitality

Hieracium pilosella

PCA analysis showed that there was no differentiation intotwo plant groups collected from sterile and non-sterile sub-strate when we consider photosynthetic parameters forM. sativa. In the case of H. pilosella, two groups of sampleswere recognized: sterile and non-sterile one (Supplement 1).

Statistical analysis of photosynthetic parameters obtainedfor H. pilosella showed interaction between plants grown onsterilized substratum/non-sterile and provided inoculationtreatment. The result of ANOVA for experimental datashowed Fv/Fm, TR0/RC, ET0/RC, RE0/RC, and PITOTAL,PIABS, DFABS, and DFTOTAL changed significantly after inoc-ulation (Fig. 5a, b). Microbial inoculation resulted in increaseof Fv/Fm parameter forH. pilosella grown on sterile substrate,except the case when H. pilosella was only co-cropped withM. sativa. Measured values of Fv/Fm were in the range of0.72–0.75. On non-sterilized substrate, Fv/Fm values were sig-nificantly lower (0.67–0.78) only when triplicate co-inoculation was provided (mycorrhiza and both bacterialstrains). Single and triplicate AMF inoculation (Myc andMyc + Bact) induced a decrease by 23–10 % of ABS/RCand 38–23 % of DI0/RC on sterile substrate; this decrease iscounterbalanced in the single or dual inoculation withdiazotrophs (Bact). On sterilized substrate, AMF single and

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Fig. 3 Relative arbuscular richness (A%) evaluated for a Hieraciumpilosella (Hp), b Medicago sativa (Ms), when plants were grown on NSnon-sterilized substrate, S dry heat sterilized substrate; plants wereinoculated with M Rhizophagus irregularis, A Azospirillum sp., NNostoc edaphicum; different letters beside the values indicatestatistically significant difference (P<0.05)

19148 Environ Sci Pollut Res (2015) 22:19142–19156

triple inoculation (Myc and Myc + Bact) significantly in-creased PIABS by (45–90 %) and PITOTAL by (25–50 %) ofH. pilosella, while double inoculation with N2-fixers caused adecrease of PI parameters (Fig. 5b). Inoculation with AMFand both N2-fixers increased DFABS and DFTOTAL by 25–30 % compared to control (Fig. 5b). On NS substrate, singleinoculation with AMF (Myc) and triplicate inoculation withmycorrhiza and diazotrophs (Myc + Bact) decreased bothABS/RC (20 %) and TR0/RC parameter (40 %). Figure 5bdemonstrates that single inoculation with AMF (Myc) in-creased PIABS by 25 %, and the triplicate inoculation withAMF inoculation and both diazotrophs (Myc + Bact) in-creased PITOTAL around 10 % (Fig. 5). Single mycorrizal in-oculation (Myc) increased DFTOTAL of H. pilosella (Fig. 5).

Medicago sativa

The result of ANOVA evaluated for alfalfa showed no in-teractions observed between sterilization procedure (plantsgrown on sterile or non-sterile substrate) and provided mi-crobial inoculation. Microbial inoculation did not causeany significant changes of Fv/Fm parameter in alfalfa sam-ple, neither when plants were grown on sterile and non-sterile substrate. Fv/Fm values obtained for alfalfa were inthe range of 0.83 and 0.85. The analysis of specific energyfluxes per QA− reducing reaction center revealed differ-ences in absorption (ABS/RC), trapping (TR0/RC), reduc-tion of end acceptors at PSI electron acceptor side (ET0/RC), and in the dissipated energy flux (DI0/RC) between

controls and inoculated with microbe plants (Fig. 6). Mi-crobial inoculation affected the performance index PIABSof alfalfa. PIABS parameter increased when plants wereinoculated with AMF (Myc and Myc + Bact), while bac-terial inoculation (Bact) caused a decrease of this parame-ter (Fig. 6). Figure 6a showed that on sterile substratum, adecrease of ABS/RC, TR0/RC, and RE0/RC by 4–7 % wasobserved in the case of single inoculation with AMF (Myc)and tr ipl icate inoculat ions with mycorrhiza anddiazotrophs (Myc + Bact). Different effect was observedfor M. sativa grown on NS case, where single inoculationwith R. irregularis (Myc) increased ABS/RC, TR0/RC, andRE0/RC by 9–22 % (Fig. 6). Although, there was no sig-nificant changes in phenomenological energy flux param-eters observed in non-sterile treatment when M. sativa wasdouble inoculated with diazotrophs (Bact) and triplicatewith AMF + N2-fixers (Myc + Bact). In NS substrate, justdouble inoculation with N2-fixers (Bact) and triplicate in-oculation with AMF + diazotrophs (Myc + Bact) resultedin the decrease of PITOTAL parameter by 23–20 %. Al-though, inoculation with mycorrhizal fungi R. irregularis(Myc) caused an increase of performance indexes PITOTALby 10 %. The same results were observed for DFTOTALparameter (Fig. 6b).

Discussion

Co-cropping

The polluted sites require combination of different restorationapproaches such as intercropping, co-cropping, or pre-cropping to improve the establishment of a plant cover onmetal-rich industrial wastes (Khan 2005; Sprocati et al.2014). H. pilosella and M. sativa were selected because theirpotential ability to be used in vegetation practices on heavymetal-rich polluted sites. Clonal plants such as H. pilosellaspread horizontally within their habitat by means of stolons,rhizomes, and ramets. Connection between ramets allow fortranslocation of resources within the clone. Through a spatialdivision of labor, clonal species were able to perform specifictasks and closely co-operate by potentially independency in-cluding reproduction (Stueffer et al. 1996). Developing vege-tative reproduction system and exhibiting phenotypic plastic-ity allows H. pilosella to overcome the establishment risk andregenerate under unfavorable conditions such as dry periodsand lack of nutrients (Salzman 1985; Winkler and Stöcklin2002; Roiloa and Retuerto 2012). H. pilosella populationsusually almost exclusively depend on clonal reproduction(Bishop and Davy 1985). It has been claimed thatH. pilosellais allelopatic species (Murphy and Aarssen 1995;Murphy 2000; Jankowska et al. 2014). However, ourexperiment reveals suppression of H. pilosella by M. sativa

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Fig. 4 Number of nodules per 50 cm ofMedicago sativa roots (Ms) co-cropped with Hieracium pilosella (Hp). Number of nodules wasestimated after 3 months of growth on a Zn–Pb-rich tailing: NS non-sterilized substrate, S dry heat sterilized substrate. Plants wereinoculated with M Rhizophagus irregularis, A Azospirillum sp., NNostoc edaphicum

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rather than the reverse. Similar results were obtained forH. pilosella and Arrhenatherum elatius in colliery spoils ofnorth of France (Henn et al. 1988). The leguminous speciesM. sativa has been suggested as a good candidate for remedi-ation of contaminated soils, due to its rhizobial symbionts andability to fix atmospheric N2 (Gardea-Torresdey et al. 1998,1999; Peralta-Videa et al. 2002; Lin et al. 2007). Co-croppingdifferent species may enhance the overall capabilities of aphytoremediation. However, under unfavorable conditionslike the presence of heavy metals, co-cropping ofH. pilosella (non-fixers) and M. sativa (legume) caused anegative effect regarding H. pilosella growth and perfor-mance (Figs. 1a and 5a). Environmental conditions maydisturb the benefits from co-cropping of non-fixing plantswith legumes, which under certain conditions, like a pres-ence of heavy metals, might exhibit a limited capacity tofix nitrogen. On the other hand, potential benefits gainedby a neighboring non-fixer plant will strongly depend ontheir capacity to use effectively the extra N input(Temperton et al. 2007). This could be caused by the com-petition for other macroelements and microelements aswell as for water and light (Tilman et al. 1997).

Substrate sterilization procedure

In general, different microbial populations may establishthemselves under the influence of root exudates and bioavail-ability of essential nutrients. However, the presence of toxicmetals strongly determines the size and composition of micro-bial populations in the rhizosphere (Bever et al. 2010). Differ-ent soil sterilization procedures are proposed in biologicalresearches concerning microbial influence or heavy metal im-pact on plant growth. Without the assurance of sterile condi-tions, obtained metal data sets can be misinterpreted as sorp-tion of metals to solid substrate or losses due to biologicalactivities. Of concern is the need to eliminate biological activ-ity in a soil sample where single interactions between plantsand selected microorganisms are investigated. Since manysterilization methods can dramatically change physical andchemical properties of the soil, it is therefore important tochoose the least destructive. Where chemical stability is re-quired, air-dry sterilization rather than moisture is recom-mended (Lotrario et al. 1995; Trevors 1996; Egli et al. 2006).

However, our results indicate that even dry heat steriliza-tion disturbed primarily established relationships between

H. pilosella – Sterile (S)

H. pilosella – Non-Sterile (NS)

Hp+Ms Hp+Ms +Bact Hp+Ms +Myc Hp+Ms+Myc+Bact

nd

Fig. 5 Deviation of the fluxes as expressed relative to control plantsHieracium pilosella. The values are expressed in percents (%) andreflect the deviation flux differences between non-inoculated plants andplants after single inoculation with the AM fungus Rhizophagusirregularis (Myc), dual inoculation with the diazotrophs bacteria:Azospirillum sp. and Nostoc edaphicum (Bact), triplicate co-inoculations with AMF fungus R. irregularis and diazotroph bacteria

(Myc + Bact), plants grown on NS non-sterilized substrate, S dry heatsterilized substrate. ABS/RC the average absorption per RC, DI0/RC thedissipated energy flux per RC, TR0/RC the specific trapping flux per RC,ET0/RC the maximal specific flux for electron transport per RC, PIABSperformance index on absorption basis, PITOTAL total performance index(PI),DFABS driving force on absorption basis,DFTOTAL total driving force

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soil-plant and plants and associated microorganisms. In ourstudies, the plants’ response to microbial inoculation was sig-nificant when the substrate was dry heat sterilized. In non-sterile substrate, the microbial communities were more com-plex from the beginning. Already established relationshipsbetween native soil microbe communities and roots in non-sterile prevent plants from metal stress. Significantly higherbiomass and dry mass production of plants growing on non-sterile substrate indicate better starting conditions. For thisreason, additional inoculation of plants growing on non-sterile substrate did not cause such significant plant responses.This sterilization appears to imbalance already established mi-crobial relationships in the soil, which is why we cannot drawthe same conclusions from S and non-sterile treatment regard-ing additional mycorrhizal and N2-fixer’s inoculations.

Arbuscular mycorrhiza interaction with N2-fixers

The presence and composition of soil microbial communitieshave been shown to have large impacts on plant–plant andplant–microbial interactions and consequently plant diversity

and composition. The rhizosphere is a specialized niche de-fined as the root–soil interface, where associated microorgan-isms, roots, and soil come together (Alford et al. 2010). Rootcolonization is a competitive process that is affected by envi-ronmental conditions such as soil moisture, soil texture andpH, organic matter content, access to the nutrients, as well asspecificity of host. Thus, microbial population is one of theessential parts of dynamic rhizosphere system that affect therhizosphere soil properties such as pH, redox potential, metalconcentration, water content, bulk density, root exudation, andall the biological transformations (Barea et al. 2002; Bais et al.2006; Bakker et al. 2013). However, unfavorable conditionson polluted sites impose the need to compete for nutrients andother resources which are limited. Therefore, individualPGPR strains exhibit specificity in sensitivity to environmen-tal parameters to promote host growth and/or suppress plantpathogens (Hibbing et al. 2010). Depending on the strainsused, they may perform a number of tasks ranging from nar-row to broad (Doornbos et al. 2012; Bakker et al. 2013).Increasing attention is focused on the interactions betweenPGPR bacteria and mycorrhizal fungi (Biró et al. 2000; Mar

Ms+Hp Ms+Hp+Bact Ms+Hp+Myc Ms+Hp+Myc+Bact

M.sativa– Sterile (S)

M.sativa– Non-Sterile (NS)

Fig. 6 Deviation of the fluxes as expressed relative to control plantMedicago sativa. The values are expressed in percents (%) and reflectthe deviation flux differences between non-inoculated plants and plantsafter single inoculation with the AM fungus Rhizophagus irregularis(Myc), dual inoculation with the diazotrophs bacteria: Azospirillum sp.and Nostoc edaphicum (Bact), tripartite co-inoculations with AMFfungus R. irregularis and diazotrophs bacteria (Myc + Bact), plants

grown on NS non-sterilized substrate, S dry heat sterilized substrate.ABS/RC the average absorption per RC, DI0/RC the dissipated energyflux per RC, TR0/RC the specific trapping flux per RC, ET0/RC themaximal specific flux for electron transport per RC, PIABS performanceindex on absorption basis, PITOTAL total performance index (PI), DFABSdriving force on absorption basis, DFTOTAL total driving force

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Vázquez et al. 2000; Tsimilli-Michael et al. 2000; Barea et al.2005a, b). Possible application of AM fungi and different soilbacteria in bioremediation processes like phytostabilizationand phytoextraction has been a great concern (Barea et al.2002, 2005a; Weyens et al. 2009). Studies show that dualinoculation AM fungus with nitrogen-fixing soil bacteria en-hanced nitrogen fixation in legumes (Bagyaraj et al. 1979;Biró et al. 2000; Tsimilli-Michael et al. 2000; Scheublinet al. 2004; Temperton et al. 2007). Azospirillum brasilenseandGlomus intrarradiceswere capable of co-existing in sugarcane roots both intracellularly and intercellularly, causingchanges in the cell wall. Sugar cane plant biomass, the numberof endophytic microorganisms, and nitrogen-fixing activityincreased with joint inoculation (Bellone and de BelloneSilvia 2012). Inoculation with native bacterial and fungalstrains ensures best performance under harsh conditions suchas high metal content. Beside AMF, diazotrophs, free-livingnitrogen-fixing bacteria strongly attributed to nitrogen fixationand cause an increase of nitrogen amount available to plants(Bethlenfalvay et al. 1982; Toro et al. 1998; Biró et al. 2000;Mar Vázquez et al. 2000; Franzini et al. 2010). In case ofM. sativa, formation of nodules would be the most desirable;however, on Zn–Pb Trzebionka tailings, nodules are rarelyfound (Turnau et al. 2012), and this was also proved duringthe present investigation as shown in case of control plantsgrown on non-sterile substratum. Nodulation is an energeti-cally costly process, and legumes balance the nitrogen de-mand with the energy expense by limiting the number of nod-ules (Kassaw et al. 2015). In addition, toxic metals present inthe substratum are known to induce morphogenic responsesand as a consequence reduce the formation of rhizobial infec-tion (Potters et al. 2007; Lafuente et al. 2010). Those metalscan also lower down the expression of several nodulationgenes (Lafuente et al. 2010). Bacterial activity and the rhizo-bial symbiosis are influenced by mycorrhiza, both qualitative-ly and quantitatively (Barea et al. 2005a, b). Our results showsthat additional inoculation with selected microbes increasednumber of nodules; however, we have to underline that totalnumber of nodules is not as important as number of active one.H. pilosella responded positively to mycorrhizal inoculationbut not to single inoculation with N2-fixing bacterial symbi-onts. On the other hand, mycorrhizal inoculation together withN2-fixers always stimulates alfalfa growth, while single aswell as double inoculation with N. edaphicum or Azospirillumsp. did not have a positive effect on shoot growth. This resultmay indicate that poor substrate such as Zn–Pb-rich tailingsused in our studies may have precluded a net benefit from themycorrhizal symbiosis more strongly than from N2-fixingbacteria. This can be explained by the fact that symbioticnitrogen fixation is very phosphorus intensive due to the highATP requirement. AM fungi increased mineral and nutrientuptake where up to 80 % of plant’s phosphorus (P) needsand 25 % of its nitrogen (N) is obtained via the fungus

(Marschner and Dell 1994; Smith et al. 2004; Govindarajuluet al. 2005; Parniske 2008). The mycorrhization percentage ofplants growing on this particular Zn–Pb tailing was shown tobe usually high (Orłowska et al. 2005). While under non-polluted conditions, usually lower percentage ofmycorrhization is efficient to obtain plant growth promotingeffect (Russo et al. 2005). On industrial tailings, this might benot enough due to involvement of fungal hyphae in sequester-ing toxic metals, what results in increased metal concentrationwithin roots which is one of the known avoidance mecha-nisms (Leyval et al. 1997). Overall, mycorrhizal inoculationwith AMF always had a positive effect on plant growth. It hasbeen proposed that the presence of their exudates low and highmolecular weight compounds including carbohydrates andorganic acids. This may create a favorable environmentaround the mycorrhizal fungal hyphae and cell surface struc-tures, which could have supported microbial growth(Scheublin et al. 2004; Toljander et al. 2007; Miransari2011). Rhizodephozition is a dynamic and extremely complexprocess, where there is a loss of reduced C for the plant andwhere the organic C pool enters the soil. This stimulates thebiological activity in the rhizosphere (Jones et al. 2009). Mi-crobial activity in soil is greatly influenced by the loss ofcarbon-containing metabolites where up to 40 % of photosyn-thetically fixed carbon is secreted into the rhizosphere by plantroots (Bais et al. 2006). In non-polluted soils, the higher Ccosts to plants of maintaining both fungal and bacterial sym-bionts may result in indirect antagonistic interactions betweensymbionts (Bethlenfalvay et al. 1982; Mortimer et al. 2008;Franzini et al. 2010). However, under unfavorable conditions,plants prefer co-inoculation strategy with AMF and N2-fixers.

Extracellular polysaccharides (EPS) were purposed byBianciotto 2001 to play a crucial role in the anchoring ofA. brasilense and R. leguminosarum and in the formation ofbiofilms on the root and the AM fungus (Bianciotto et al.2001). Biofilm microorganisms may play an essential role asmediators in the transfer of heavy metals (García-Meza et al.2005). Extracellular polymeric substances potentially act asdetoxification agents against metals, acting as metal-bindingsites. Biofilm occurrence seems to enhance complexation andimmobilization of Cr, Ni, Cu, Zn, As, and Pb. Due to theorganic matter improvement, the biofilms could also be con-ceptualized as an organic cover, which controls pH (Lukešová2001; García-Meza et al. 2006). The persistence of the photo-synthetically active forms such as cyanobacteria might resultin a natural fertilization of the tailing substratum building upan appropriate environment for further plant establishment.The importance of soil cyanobacteria in increasing soil fertil-ity, through the input of nitrogen, promotes the release ofnutrients from insoluble compounds (Maxwell 1991). Studiesconducted by Trzcińska and Pawlik-Skowrońska 2008 refer tothe cyanobacteria isolated from Zn–Pb-loaded soils, includingN. edaphicum strain. Those isolated strains are Zn-Pb-resistant

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ecotypes, which have been rarely reported, in other terrestrialenvironment (Trzcińska and Pawlik-Skowrońska 2008).N. edaphicum can utilize various inorganic and organic nitro-gen sources for growth. Those nitrogen sources are utilized inthe hierarchical order of NH4+ > NO3− > N2. This flexibilityallows Nostocales to colonize and compete as a phototroph inilluminated habitats, irrespective of the specific nitrogensource (Meeks et al. 2001). This ability seems to be importantespecially in poor nutrient environment such as waste tailing.The present studies reveal positive effect on plant growth bydual inoculation with native strain of N. edaphicum and AMfungi. This might be related to nitrogen level available forAMF symbionts. However, single inoculation withcyanobacteria or with nitrogen-fixing bacteria did not have apositive effect on plant growth. In both types of substratetreatments (S and NS), AMF colonization stimulated the for-mation of root nodules in the alfalfa roots. AMF colonizationhas been known to enhance the formation of nodules, by in-crease P which then attributed to better N uptake, because ofhigher nitrogenase-fixation activity in mycorrhizal plants(Toro et al. 1998; Andrade et al. 2004; Barea et al. 2005b;Lin et al. 2007). Also, this positive role of AMF might berelated to biofilm formation. Bacteria living in the biofilmforms have the ability to survive much harsher environmentalconditions (Costerton et al. 1995). On the other hand, dualsymbiosis formed by AMF with Rhizobium strain has beenshown to inhibit nodule development and N2-fixation ofPhaseolus vulgaris (Lin et al. 2007; Franzini et al. 2010).Given this case that the formation of root nodules may bestrongly inhibited not just by heavy metals present in the soil,but also by other microorganisms, therefore, a selection ofappropriated symbionts to specific plants and environmentalcondition is needed to improve successful phytoremediation.This may be due to the competition for nutrients betweenmixed microorganisms and the ability of one organism to dealwith heavy metals more than the other (Hudek et al. 2012).

Plant photosynthesis

The JIP test is presently widely accepted for evaluation of PSIIbehavior. Organisms exposed to stress such as high light levelor heat showed pronounced decrease of φPo=TR0/ABS,measured as Fv/Fm (Strasser et al. 2004). Plants grown underoptimal conditions show values of Fv/Fm in the range 0.79–0.85 (Maxwell and Johnson 2000; Kalaji et al. 2014). Similarvalues were observed in case of plants studied in the presentresearch. In comparison to control plants (grown alone with-out addition of inocula), microbial inoculation resulted in sig-nificant increase of Fv/Fm parameter for H. pilosella, exceptthe casewhenH. pilosellawas only co-croppedwithM. sativa.On non-sterilized substrate, Fv/Fm values were significantlylower, only when triplicate co-inoculation was provided. De-crease of Fv/Fm already suggests negative effect of inoculation

on photosynthesis functioning. M. sativa did not exhibit anychanges regarding Fv/Fm parameters. Previous studies indicat-ed that performance indexes (PITOTAL, PIABS) and drivingforces (DFTOTAL, DFABS) are sensitive measures of plant re-sponses to different kinds of environmental stresses such asirradiance, drought, heat, salt, biotic-stressed, and exposure toheavy metals (Tsimilli-Michael et al. 2000; Strasser et al.2000; Strauss et al. 2006; Christen et al. 2007; Yusuf et al.2010; Kalaji et al. 2011; Oukarroum et al. 2014). Therefore,these parameters are consistent to evaluate the plant perfor-mance, especially when plants were exposed to the stress(Strasser et al. 2000; Strauss et al. 2006). Findings that mi-crobes can cause a stress have been also reported earlier(Tsimilli-Michael et al. 2000; Tsimilli-Michael and Strasser2002). Similarly to abovementioned parameters, the testshowed differences in (ABS/RC), trapping (TR0/RC), and re-duction of end acceptors at PSI electron acceptor side (ET0/RC) and the dissipated energy flux (DI0/RC) between controlsand plants inoculated with microbes as was shown in Tsimilli-Michael et al. 2000 and Strasser et al. 2004. ABS/RC thatgives the total absorption of chlorophylls in PSII antennaeper reaction centers is a good measure for average functionalantenna size (Tsimilli-Michael et al. 2000). Higher values ofABS/RC together with significant increase in DI0/RC ob-served in control plants (M. sativa and H. pilosella) could beconsidered as indicators of photoinhibitory damages to PSIIcomplexes (Force et al. 2003). Co-cropping system that in-cludes M. sativa and H. pilosella without microbial inocula-tion is not sufficient for successful remediation. Under labo-ratory conditions, plants are not exposed to such extremestresses as the one observed on industrial wastes. Therefore,the chances for plant survival are even lower. The use ofmicrobes visibly attenuates negative effect of co-croppingand additionally can be useful for nutrient cycling. Figures 6and 5 indicate similar trends in plant behavior depending onthe use of substrate sterilization procedure. Under sterile con-ditions, both plants exhibited higher PI parameters when tripleinoculation was performed. If substrate was not sterilized, theaddition of bacterial inoculation had evenmore negative effecton plant performance. As shown above, JIP test provides auseful tool for evaluation of the effectiveness of microbialinoculation that has to be taken into account, especially whenphytoremediation of polluted site has to be optimized.

Conclusions

Future bioremediation research should strive toward an im-proved understanding of the functional mechanisms behindsuch microbial interactions, so that optimized combinationsof microorganisms can be applied as effective inoculants with-in sustainable remediation systems. Appropriate bioremedia-tion practices may include these inoculates to obtain a

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beneficial effect on plant establishment on heavy metal-polluted sites. Our experimental tests confirm that both nega-tive and positive feedbacks occur between plants and micro-bial communities. An improved understanding of the interac-tions taking place in the rhizosphere will help to translate theresults of simplified experiments into field application. Thecharacteristics of a microbial network in laboratory conditionscould help to design specific remediation strategy on metal-rich industrial wastes.

Acknowledgments This work was supported by the Foundation forPolish Science, International PhD Projects Programme co-financed bythe EU European Regional Development Fund (MPD/2009-3/5) and byproject MAESTRO contract no. 2011/02/A/NZ9/00137. Funding wasalso provided by the Małopolskie Centre of Entrepreneurship-Programme DOCTUS (ZS. 4112-129/2010). The authors would like tothank Prof. Konrad Wołowski (Institute of Botany Polish Academy ofScience, Krakow) for identification of cyanobacteria and Prof. ZbigniewMiszalski for constructive comments. Special thanks to Dr. RafałWażnyfrom TheMalopolska Center of Biotechnology for his help in preparationof materials for analysis.

Open Access This article is distributed under the terms of the CreativeCommons Attr ibution 4.0 International License (http: / /creativecommons.org/licenses/by/4.0/), which permits unrestricteduse, distribution, and reproduction in any medium, provided you giveappropriate credit to the original author(s) and the source, provide a linkto the Creative Commons license, and indicate if changes were made.

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